Platelet Reactive Oxygen Species Generation Research Articles

Platelets as Potential Non-Traditional Cardiovascular Risk Factor-Analysis Performed in Healthy Donors.

Abnormal lipid profile, increased glucose level, and elevated body weight are traditional cardiometabolic risk factors; however, the role of platelets in the development of cardiovascular disease (CVD) is increasingly being highlighted. The aim of this study was to select platelet-related parameters (non-genetic molecular and routine laboratory measurements) that may be associated with increased cardiovascular risk among healthy populations. We evaluated the level of platelet indices, platelet-based inflammatory markers, platelet reactivity parameters, and platelet reactive oxygen species (ROS) generation in relation to selected cardiometabolic risk factors. We noted the association between total cholesterol and LDL cholesterol with platelet aggregation and platelet ROS generation. We found the relationship between triglycerides, glucose, and body mass index with the relatively new multi-inflammatory indices (MII-1 and MII-3). Moreover, we noticed that the mean platelet volume-to-lymphocyte ratio in healthy subjects is not a good source of information about platelets and inflammation. We also highlighted that platelet-to-HDL-cholesterol ratio may be a promising prognostic cardiometabolic indicator. The association between platelet-related (especially molecular) and cardiometabolic parameters requires further research. However, the goal of this study was to shed light on the consideration of platelets as a non-traditional cardiovascular risk factor and a crucial element in identifying individuals at high-risk of developing CVD in the future.

Platelet CD40L Expression Response to Mixing of pRBCs and Washed Platelets but no Causality Association between Platelet ROS Generation and CD40L Expression: An In Vitro Study.

Platelets play a role in transfusion reaction via reactive oxygen species (ROS) generation and CD40 ligand (CD40L) expression. In this study, we aimed to test the hypothesis that the mixing of packed red blood cells (pRBCs) and washed platelets has a causal effect on platelet ROS generation and CD40L expression. Thus, a better understanding of this causality relationship may help interrupt the chain of events and avoid an uncontrollable transfusion reaction. We simulated transfusion in vitro by mixing pRBCs and washed platelets. Donor cross-matched stored pRBCs) from our blood bank and recipient whole blood from patients undergoing coronary artery bypass graft surgery prepared into washed platelets were used. Briefly, donor pRBCs were added to washed recipient platelets to form 1%, 5%, or 10% (v/v) mixtures. The mixed blood sample was used to determine platelet ROS generation (dichlorofluorescein fluorescence levels) and CD40L expression. The effect of antioxidants (20 mM glutamine and 20 mM dipeptiven) on ROS generation and CD40L expression was also evaluated. Platelet ROS generation was not significantly associated with the mixing of pRBCs and washed platelets (p = 0.755), glutamine treatment (p = 0.800), or dipeptiven treatment (p = 0.711). The expression of CD40L by platelets increased significantly (p < 0.001), and no significant difference was noted after treatment with glutamine (p = 0.560) or dipeptiven (p = 0.618). We observed that the mixing pRBCs and washed platelets had no effect via ROS, whereas CD40L could directly induce transfusion reactions. Furthermore, platelets did not causally express ROS or CD40L after being mixed with pRBCs. Although antioxidants are more accessible than anti-CD40L antibodies, platelet ROS may not serve as a therapeutic target for antioxidants. Nevertheless, CD40L expression may be a valuable therapeutic target for managing transfusion reactions.

Shear and Integrin Outside-In Signaling Activate NADPH-Oxidase 2 to Promote Platelet Activation.

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Platelet Oxidative Stress and its Relationship with Cardiovascular Diseases in Type 2 Diabetes Mellitus Patients.

Enhanced platelet activation and thrombosis are linked to various cardiovascular diseases (CVD). Among other mechanisms, oxidative stress seems to play a pivotal role in platelet hyperactivity. Indeed, upon stimulation by physiological agonists, human platelets generate and release several types of reactive oxygen species (ROS) such as O2 -, H2O2 or OH-, further amplifying the platelet activation response via various signalling pathways, including, formation of isoprostanes, Ca2+ mobilization and NO inactivation. Furthermore, excessive platelet ROS generation, incorporation of free radicals from environment and/or depletion of antioxidants induce pro-oxidant, pro-inflammatory and platelet hyperaggregability effects, leading to the incidence of cardiovascular events. Here, we review the current knowledge regarding the effect of oxidative stress on platelet signaling pathways and its implication in CVD such as type 2 diabetes mellitus. We also summarize the role of natural antioxidants included in vegetables, fruits and medicinal herbs in reducing platelet function via an oxidative stress-mediated mechanism.

Platelet granule release is associated with reactive oxygen species generation during platelet storage: A direct link between platelet pro-inflammatory and oxidation states

BackgroundUpon platelet stimulation with agonists, reactive oxygen species (ROS) generation enhances platelet activation and granule release. Whether ROS generation during platelet storage could be directly correlated with the expression of proinflammatory molecules and granule release has been investigated in this study. Material and methodPRP-platelet concentrates were subjected to flowcytometry analysis to assess the expression of platelet activation marker, P-selectin and CD40L during storage. Intracellular ROS generation was also detected in platelet by flowcytometry using dihydrorhodamine (DHR) 123. Through the dual staining, ROS production was analyzed in either P-selectin positive or negative populations. ResultsROS formation in platelet population was significantly increased by either TRAP (a potent agonist that induces granule release) or PMA (a classic inducer of ROS generation), while the effects of each agonists on P-selectin expression and ROS generation in platelets were comparable. Platelet storage was also associated with the increasing levels of ROS (day 0 vs. day 5; p<0.001) while this increasing pattern was directly correlated with the either expressed P-selectin or CD40L. In addition, in 5 day-stored platelets, samples with ROS levels above 40% showed significantly higher levels of P-selectin and CD40L expression. P-selectin negative population of platelet did not show significant amount of ROS. ConclusionOur data demonstrated decreased levels of important platelet pro-inflammatory molecules in stored platelets with lower levels of intraplatelet ROS. However, whether quenching of ROS generation during platelet storage can attenuate adverse transfusion reactions raised by platelet pro-inflammatory status is required to be further studied.

RhoA and Rac1 GTPases Differentially Regulate Agonist-Receptor Mediated Reactive Oxygen Species Generation in Platelets.

Agonist induced generation of reactive oxygen species (ROS) by NADPH oxidases (NOX) enhances platelet aggregation and hence the risk of thrombosis. RhoA and Rac1 GTPases are involved in ROS generation by NOX in a variety of cells, but their roles in platelet ROS production remain unclear. In this study we used platelets from RhoA and Rac1 conditional knockout mice as well as human platelets treated with Rhosin and NSC23767, rationally designed small molecule inhibitors of RhoA and Rac GTPases, respectively, to better define the contributions of RhoA and Rac1 signaling to ROS generation and platelet activation. Treatment of platelets with Rhosin inhibited: (a) U46619 induced activation of RhoA; (b) phosphorylation of p47phox, a critical component of NOX; (c) U46619 or thrombin induced ROS generation; (d) phosphorylation of myosin light chain (MLC); (e) platelet shape change; (f) platelet spreading on immobilized fibrinogen; and (g) release of P-selectin, secretion of ATP and aggregation. Conditional deletion of RhoA or Rac1 gene inhibited thrombin induced ROS generation in platelets. Addition of Y27632, a RhoA inhibitor, NSC23766 or Phox-I, an inhibitor of Rac1-p67phox interaction, to human platelets blocked thrombin induced ROS generation. These data suggest that: (a) RhoA/ROCK/p47phox signaling axis promotes ROS production that, at least in part, contributes to platelet activation in conjunction with or independent of the RhoA/ROCK mediated phosphorylation of MLC; and (b) RhoA and Rac1 differentially regulate ROS generation by inhibiting phosphorylation of p47phox and Rac1-p67phox interaction, respectively.

Abstract 58: Differential Signaling by CRP-Rac1-NOX1 and Thrombin-Rac1-NOX2 Axes Regulates Ros Generation and Platelet Activation

Agonist induced generation of reactive oxygen species (ROS) enhances platelet aggregation and hence the risk of thrombosis. Rac1 GTPase has been shown to be critical in ROS generation by both NOX1 and NOX2. However, its role in agonist induced ROS generation in platelets remains to be determined. We have shown previously that gene targeting or inhibition of Rac1 GTPase inhibits platelet secretion and aggregation. In this study we tested our hypothesis that Rac1 regulates platelet activation, at least in part, by regulating ROS generation through the NOX enzymes by using Rac1 and NOX2 gene targeted mice and/or pharmacologic inhibitors. ROS generation was determined by flow cytometry in dcf-da loaded washed platelets. Collagen-related peptide (CRP) or thrombin induced ROS generation in a time- and a concentration-dependent manner. A two minute pre-incubation of platelets with NSC23766 (3-30 μM), a Rac GTPases inhibitor, or Phox-I (1-10 μM), an inhibitor of NOX2 that blocks Rac1-p67 phox interaction, diminished CRP or thrombin induced ROS generation, ATP secretion and aggregation. However, treatment of platelets with NSC23766 or Phox-I did not inhibit phosphorylation of p47 phox , a critical subunit of NOX. Addition of CRP or thrombin induced significantly less ROS generation in platelets from Rac1 -/- , as compared to Rac1 +/+ , mice. Addition of CRP generated significantly less ROS and exhibited diminished aggregation in NOX2 -/- platelets than NOX2 +/+ platelets. A two minute incubation of platelets with ML171 (0.1 -1.0 μM), a selective inhibitor of NOX1, inhibited CRP induced but not thrombin induced ROS generation, ATP secretion and aggregation. Addition of a combination of ML171 and NSC23766, Phox-I and NSC23766 or ML171and Phox-I to platelets did not result in additive inhibitory effect on CRP induced ROS generation. Together, these results show for the first time that Rac1 GTPase: (a) is essential for agonist induced ROS generation in platelets; (b) inhibits ROS generation by inhibiting Rac1-p67 phox interaction but not activation of p47 phox ; (c) is involved in ROS generation by both NOX1 and NOX2. The differential signaling of CRP activated Rac1-NOX1-ROS and thrombin-stimulated Rac1-NOX2-ROS play an important role in platelet secretion and aggregation.

RhoA and Rac1 Gtpases Differentially Regulate Agonist-Receptor Mediated ROS Generation in Platelets

Agonist induced generation of reactive oxygen species (ROS) including superoxide anion (O-2) and hydrogen peroxide (H2O2) enhance platelet aggregation and hence the risk of thrombosis. Although diverse biochemical reactions contribute to ROS generation, NADPH oxidases (NOX) have emerged as critical sources of agonist induced ROS generation in platelets. Previous studies have shown that small GTPases Rac1 and RhoA are involved in NOX activation. Rac GTPase activates NOX by directly binding to NOX as well as by interacting with p67phox to promote its binding to NOX (Physiol Rev 87: 245–313, 2007), whereas RhoA triggers ROS generation via the ROCK/p38MAPK cascade mediated phosphorylation of p47phox, a critical component of the NOX complex, (Exp Mol Med 37:575-87, 2005). To date, however, the roles of Rac1 and RhoA in platelet ROS production remain unclear. This study was conducted to define the contributions of Rac1- and RhoA- signaling to ROS generation and platelet function. ROS generation was quantified by flow cytometry in dcf-da (10 µM) loaded washed platelets. Thrombin has been shown to generate ROS in human platelets (Blood 106: 2757-2760, 2005). In this study we confirmed that platelets stimulated with thrombin generate ROS in a time- and a concentration- dependent manner. Addition of thrombin to human platelets pre-treated with NSC23766, a Rac-specific inhibitor, or murine platelets with Rac1 gene deletion, produced significantly less ROS than the matching control samples. Further, Phox-I, a pharmacologic inhibitor of Rac-p67phox interaction (Chem Biol 19: 228-24, 2012), potently suppressed thrombin induced ROS production, indicating that a Rac1-p67phox signaling axis is involved in thrombin mediated ROS production. Separately, treatment of washed human platelets with a RhoA specific inhibitor, Rhosin (Chem Biol 19:699-710, 2012) resulted in: (a) inhibition of the U46619, a stable analog of TXA2, induced activation of RhoA, but not that of Rac1or Cdc42; (b) U46619 induced phosphorylation p38MAPK and p47phox; and (c) U46619 or thrombin induced ROS generation. We further investigated the role of RhoA/ROCK/p38MAPK in ROS production by using platelets from RhoA-/- mice, Y27632 (a ROCK inhibitor) and SB203580 (a p38MAPK inhibitor). RhoA-/- platelets or human platelets treated with Y27632 or SB203580 exhibited significantly diminished ROS generation in response to thrombin. Next, we investigated the physiological effects of Rhosin on platelet activation. A pre-incubation of washed human platelets with Rhosin inhibited U46619 or thrombin induced platelet shape change, release of P-selectin, secretion of ATP and aggregation. The anti-platelet effects of Rhosin were reversible as washing of platelets after incubation with Rhosin abolished the inhibitory effect of Rhosin on platelet aggregation. These results suggest that (a) RhoA signaling, through ROCK/MAPK/p47phox activation, leads to ROS generation and platelet activation in conjunction with or independent of the RhoA/ROCK mediated phosphorylation of MLC, and (b) Rac1 and RhoA differentially regulate platelet ROS generation by directly binding to NOX, promoting binding of p67phox to NOX and by phosphorylation of p47phox, respectively. DisclosuresNo relevant conflicts of interest to declare.

Abstract 196: NADPH Oxidase Isoforms NOX1 and NOX2 Differentially Regulate GPVI- and Non-GPVI-Dependent ROS Generation and Platelet Activation

Reactive oxygen species (ROS) play an important role in platelet activation. Although diverse biochemical reactions contribute to ROS generation, NADPH oxidases (NOX) have emerged as critical sources of agonist induced ROS generation in platelets. Platelets express two NOX isoforms, NOX1 and NOX2. In this study we investigated the effects of ML171 (0.1-1 μM), a selective NOX1 inhibitor (ACS Chem Biol. 5, 981-993, 2010), and Phox-I (1-10 μM), a rationally designed small molecule inhibitor of NOX2 (Chem. Biol. 19, 699-710, 2012), to define the roles of NOX1 and NOX2 in ROS-mediated platelet activation by diverse agonists. A two-minute pre-incubation of washed human platelets with ML171 or Phox-I inhibited CRP, a GPVI agonist, induced ROS generation, ATP secretion and platelet aggregation. In contrast, only platelets treated with Phox-I, but not ML171, inhibited CRP induced binding of PAC-1 to platelets and expression of P-selectin. Treatment of platelets with Phox-I, but not ML-171, inhibited thrombin or U46619, a stable analog of TXA 2 , induced ROS generation, P-selectin expression, ATP secretion and aggregation. Both Phox-I and ML171 inhibited CRP induced phosphorylation of myosin light chain (MLC) and Akt. However, only Phox-I inhibited thrombin induced phosphorylation of MLC or Akt. Treatment of platelets with Phox-I or ML171 only minimally or partially inhibited CRP induced phosphorylation of p38-MAPK or ERK. In other experiments, incubation of platelets with varying concentrations of both ML171 and Phox-I did not exhibit additive effect on CRP induced ROS generation. These results imply that ROS generation by these NOX isoforms may be sequential. These data suggest that NOX1 is specifically involved in GPVI mediated, whereas NOX2 is involved in GPVI- and non-GPVI-dependent, signals in ROS generation and platelet activation.

Abstract 20: RhoA GTPase Regulates Reactive Oxygen Species--Mediated Platelet Activation

Rho family GTPases and reactive oxygen species (ROS) are known to be involved in platelet activation. Although Rac1, a Rho member, has been shown to be critical for ROS generation by NADPH oxidase (NOX) and platelet activation, RhoA, the founding member of the Rho family, has not been suspected in ROS generation and associated platelet activation. In this study we investigated the effects of Rhosin, a rationally designed small molecule that specifically binds to RhoA at the site required for its activation by Rho-GEF and thereby prevents activation of RhoA (Chem. Biol. 19, 699-710, 2012) on ROS generation and platelet activation to better understand the role of RhoA in platelet signaling. Addition of Rhosin to washed human platelets two minutes prior to stimulation with U46619, a stable analog of TXA 2 , inhibited (a) activation of RhoA but not of Rac1 or Cdc42, (b) ROS generation, and (c) phosphorylation of p47 phox , a critical component of the NOX complex. In other experiments, U46619 induced ROS generation in platelets from RhoA +/+ but not in RhoA -/- mice. Treatment of platelets with Rhosin inhibited U46619-induced (a) phosphorylation of myosin light chain (MLC) in the presence or absence of aspirin and apyrase, (b) platelet shape change, filopodia formation and spreading on immobilized fibrinogen, (c) release of P-selectin, secretion of ATP and aggregation, and (d) phosphorylation of ERK and p 38 -MAPK, and Akt. These findings suggest that RhoA induced ROS generation contributes to platelet activation via phosphorylation of p 38 -MAPK, ERK and Akt. Rhosin also inhibited thrombin induced ROS generation, platelet shape change, secretion, aggregation and clot retraction. Washing of platelets after incubation with Rhosin abolished the inhibitory effects of Rhosin on platelet activation. Administration of Rhosin to mice prolonged tail bleeding times. These data suggest that: (a) agonist-RhoA/ROCK mediated phosphorylation of p47 phox plays a critical role in ROS generation which in turn induces platelet activation in conjunction with or independent of the RhoA/ROCK mediated phosphorylation of MLC; and (b) targeting RhoA by rationally designed small molecule inhibitors offers a novel approach for developing safer and more effective antithrombotic agent.

Urtica dioica extract reduces platelet hyperaggregability in type 2 diabetes mellitus by inhibition of oxidant production, Ca&lt;sup&gt;2+&lt;/sup&gt; mobilization and protein tyrosine phosphorylation

Platelet hyperaggregability is involved in the pathogenesis of type 2 diabetes mellitus. Thrombin-evoked platelet aggregation includes the activation of several intracellular pathways, including endogenous generation of reactive oxygen species (ROS), Ca2+ mobilization and protein tyrosine phosphorylation. Here we show that crude aqueous extract from Urtica dioica reduces thrombin-evoked aggregation in platelets from healthy donors and diabetics, in a concentration-dependent manner. U. dioica extract showed a potent antioxidant activity and prevented thrombin-evoked ROS generation in platelets from healthy and diabetic donors. Treatment with U. dioica extract reduced Ca2+ entry induced by thrombin or selective depletion of the two Ca2+ stores in platelets (without altering Ca2+ release), reduced protein tyrosine phosphorylation and reversed the abnormal Ca2+ mobilization and tyrosine phosphorylation in platelets from diabetics. We conclude that extract from U. dioica shows antiaggregant actions and might be used for the treatment and/or prevention of cardiovascular complications associated with type 2 diabetes mellitus.

The Effect of Dipyridamole on Vascular Cell-Derived Reactive Oxygen Species

Platelet and vascular stimulation leads to release of reactive oxygen species (ROS) that are known to influence vascular reactivity and thrombosis. Dipyridamole is a vasodilator and platelet inhibitor that has previously been shown to have direct antioxidant properties. The antioxidant effects of dipyridamole on vascular cell-derived ROS are not known; therefore, dipyridamole was incubated with endothelial cells and platelets and cellular redox status and release of endogenous ROS were assessed. Dipyridamole decreased intracellular basal ROS generation from endothelial cells as measured by DCFDA (2',7'-dichlorodihydrofluorescein diacetate) oxidation. Incubation of endothelial cells with dipyridamole also attenuated t-butylhydroperoxide-induced oxidative stress. Using a redox-sensitive fluorescent dye, dipyridamole improved cellular activity after treatment with t-butylhydroperoxide. Incubation with dipyridamole did not alter platelet release of nitric oxide or hydrogen peroxide but significantly attenuated superoxide release. Using flow cytometry and confocal microscopy, dipyridamole decreased platelet ROS generation. Dipyridamole also suppressed platelet-soluble CD40 ligand release. In summary, at therapeutically relevant concentrations, dipyridamole suppresses the formation of ROS in platelets and endothelial cells and improves cellular redox status. These data suggest that the redox-dependent properties of dipyridamole have a direct effect on vascular cells.